Expression of listeriolysin O and ActA by intracellular and extracellular Listeria monocytogenes

Listeria monocytogenes requires listeriolysin O (LLO) and ActA, the product s of hly and actA, respectively, to establish a productive intracellular in fection, LLO is essential for vacuolar lysis and entry into the cytosol, wh ile ActA is required for bacterial spread to adjacent cells. We have used a transcriptional reporter gene system to compare the expression of actA and hly during intracellular growth to that during growth in broth cultures. T he hly and actA genes were transcriptionally fused to Escherichia coil lacZ and Bacillus pumilus cat-86 (cat), and the fusions were integrated in sing le copies into the L. monocytogenes chromosome. A chloramphenicol resistanc e assay indicated that the hly fusion but not the actA fusion was significa ntly activated in Luria-Bertani (LB) broth, and this finding correlated wit h LLO and ActA le,els detectable in broth cultures. Quantitation of promote r activity on the basis of beta-galactosidase activity revealed up to 10-fo ld-higher level of expression of the hly fusion relative to the actA fusion in LB broth. In contrast, both fusions were active in the cytosol of J774 cells, and the activity of the actA fusion was approximately 3-fold higher than that of the hly fusion under these conditions, However, quantitative i mmunoprecipitation of ActA and LLO from infected J774 cells demonstrated ap proximately 70-fold more cytosolic ActA than cytosolic LLO, Finally, in com parison to induction in broth cultures, actA was highly induced (226-fold) and hly was moderately induced (20-fold) in J774 cells. Collectively, these results indicate that actA and hly are differentially regulated in respons e to the growth environment and that both genes are preferentially expresse d during intracellular growth. Further, while the lower level of production of ActA than of LLO in broth can be accounted for by transcriptional regul ation, the relative abundance of intracellular ActA compared to that of int racellular LLO is a function of additional, possibly host-mediated, factors .